Monoamine oxidase (MAO) (E C 1.4.3.4.) may probably be a source of hydrogen peroxide which is needed in the biosynthesis of thyroid hormones. The presence of two functionally different forms of the enzyme with distinct sensitivity to inhibitors and preferred substrates has been described in several tissues. In this work the effect of two selective MAO inhibitors on iodotyrosine formation induced by those monoamines that are MAO substrates was studied in order to determine which forms of the enzyme participate in the hydrogen peroxide generation. Iodotyrosine formation was measured in a bovine thyroid subcellular fraction (30,000xg) preincubated with the inhibitors 15 min at 25 C, in a ¹³¹I-KI and tyrosine excess. MAO substrates (0.1 mM) 5-hydroxytryptamine (5-HT) (preferred of A form), tyramine (both forms) and beta-phenylethylamine (PEA) (B form) and MAO inhibitors (10^-4 M-10^-12M) clorgyline (selective of MAO A) and deprenyl (MAO B) were used. The iodotyrosine formation induced by the three MAO substrates was markedly inhibited from low concentrations of clorgyline whereas it was hardly reduced by deprenyl. These results showed that the iodotyrosine formation was strongly sensitive to clorgyline towards the three MAO substrates. PEA, usually classified as a substrate for the B form, was metabolized by MAO A. Peroxidase activity in the particulate fraction assayed in the presence of each one of the MAO inhibitors was not modified. These studies indicate that hydrogen peroxide generated by the amine metabolism mainly by the A form of MAO may be used in the iodination process in the bovine thyroid particulate fraction. Selective inhibitors of the A form but not those of the B form of the enzyme would be able to inhibit the iodination in these tissue. The present findings are in agreement with our previous reports which indicated that the bovine thyroid tissue is highly enriched with the A form of MAO.